RasGRP1 is a Ras guanine nucleotide exchange factor (GEF), and an essential regulator of lymphocyte receptor signaling. Aberrant expression of RasGRP1 results in defective positive thymocyte selection in mice. Moreover, recent reports describe RasGRP1 deficient patients that suffer from recurrent infections and autoimmunity. It is unknown how RasGRP1 levels are regulated and how aberrant expression contributes to autoimmunity.
We demonstrated that T cell positive selection was mildly impaired in rasgrp1+/- C57/B6 mice and severely impaired in rasgrp1-/- C57/B6 mice, which resulted in elevated levels of serum antinuclear antibodies, indicating that loss of RasGRP1 expression causes inflammatory disease and the severity of the phenotype is inversely correlated with RasGRP1 expression levels.
In patients with autoimmunity, we only detected decreased RASGRP1 mRNA levels in peripheral CD4+ T cells during active inflammation.
Next, by analyzing H3K27 acetylation profiles, we identified two 'autoimmunity hotspots' in the RasGRP1 gene, that showed distinct H3K27 acetylation in CD4+ T cells, and contained autoimmunity-associated SNPs. CRISPR-Cas9 editing of the 'autoimmunity hotspot' upstream of the RasGRP1 promoter, resulted in lower RasGRP1 expression and Ras-MAPK signaling, proving its enhancer function.
Furthermore, with affinity-purification mass spectrometry, we showed that mutation of this hotspot led to decreased binding of transcription factors Runx1, and ZBTB9.
In conclusion, we show for the first time how expression of RasGRP1 is regulated, and that proper regulation of RASGRP1 expression is vital to prevent inflammatory disease.